S118: IDENTIFICATION OF DIRECT TRANSCRIPTIONAL TARGET GENES OF NUP98-KDM5A REVEALS REGULATORY NETWORKS IN ACUTE MYELOID LEUKEMIA
Authors: S. Troester, J. Schmoellerl, T. Eder, G. Manhart, G. Winter, J. Zuber, F. Grebien
Journal: HemaSphere (2022)

Background: Oncogenic fusion proteins involving the Nucleoporin 98 (NUP98) gene are recurrently found in acute myeloid leukemia (AML) with a particular prevalence in pediatric patients. A chromosomal rearrangement resulting in the fusion of NUP98 to the gene encoding the lysine-specific demethylase 5A (KDM5A) is the most frequent NUP98-fusion in infant leukemia and is associated with particularly poor prognosis. The urgent need for the development of tailored treatments requires a better understanding of the effects of NUP98-KDM5A on the deregulation of gene expression programs. Although it has been shown that oncogenic NUP98-fusion proteins act as transcriptional regulators, it is unclear if and how NUP98-KDM5A directly regulates gene expression to drive leukemia. Aims: In this study, we aimed to identify immediate critical effectors of the NUP98-KDM5A fusion protein and to characterize the transcriptional programs through which they regulate the development and maintenance of NUP98-KDM5A-driven AML. Methods: We conducted a genome-scale CRISPR/Cas9 loss-of-function screen in a NUP98-KDM5A-driven murine AML cell line to unravel functional genetic dependencies that could be exploited to target leukemia cells. In parallel, we developed a new model for degradation tag (dTAG)-mediated ligand-induced degradation of the NUP98-KDM5A protein to gain a detailed understanding of direct transcriptional effects of NUP98-fusion-dependent gene regulation. We used this model to measure immediate changes in transcription upon acute NUP98-KDM5A degradation by nascent RNA-seq (SLAM-seq). An inducible shRNA system was used to assess the requirements of direct NUP98-KDM5A target genes for leukemia cell growth and to measure global gene expression changes by RNA-seq. Results: Analysis of the CRISPR/Cas9 screen identified 4105 genes that are required for the proliferation and survival of NUP98-KDM5A-driven AML cells. Complete loss of the dTAG-NUP98-KDM5A fusion protein was achieved within one hour after ligand addition, resulting in cell cycle arrest, terminal differentiation and apoptosis of leukemia cells. Global analysis of nascent mRNA expression by SLAM-seq revealed 45 immediate NUP98-KDM5A target genes, as their transcription was significantly downregulated upon fusion protein degradation. Among these genes, 12 were classified as essential factors for NUP98-KDM5A cell growth from the CRISPR/Cas9 screen. This list included known target genes of NUP98-fusion proteins, such as members of the Hoxa gene cluster, but also other transcription factors, enzymes and RNA binding proteins. shRNA-mediated knockdown of the 12 candidate genes confirmed their essentiality for NUP98-KDM5A leukemia cell proliferation. Through RNA-seq studies, we found that the knockdown of a small subset among the 12 candidate genes was able to recapitulate global patterns of gene deregulation that are induced by NUP98-KDM5A knockdown. Summary/Conclusion: Using a combination of CRISPR/Cas9 screening and ligand-induced degradation, we identified direct transcriptional target genes of NUP98-KDM5A that are functionally essential in AML. Multi-layered investigations of the interplay between the members of this small network of genes using a variety of models including primary patient samples will allow us to further dissect their role in the regulation of aberrant gene expression in NUP98-KDM5A-expressing leukemia cells and might identify novel therapeutic targets.